The plasma membranes of cells present a barrier to passage of many useful therapeutic agents. In general, a drug must be freely soluble in both the aqueous compartments of the body and the lipid layers through which it must pass, in order to enter cells. Highly charged molecules in particular experience difficulty in passing across membranes. Many therapeutic macromolecules such as peptides and oligonucleotides are also particularly intractable to transmembrane transport. Thus, while biotechnology has made available a greater number of potentially valuable therapeutics, bioavailability considerations often hinder their medicinal utility. There is therefore a need for reliable means of transporting drugs, and particularly macromolecules, into cells.
Heretofore, a number of transporter molecules have been proposed to escort molecules across biological membranes. Ryser et al. (1979) teaches the use of high molecular weight polymers of lysine for increasing transport of various molecules across cellular membranes, with very high molecular weights being preferred. Although the authors contemplated polymers of other positively charged residues such as ornithine and arginine, operativity of such polymers was not shown.
Frankel et al. (1991) reported that conjugating selected molecules to the tat protein of HIV can increase cellular uptake of those molecules. However, use of the tat protein has certain disadvantages, including unfavorable aggregation and insolubility properties.
Barsoum et al. (1994) and Fawell et al. (1994) proposed using shorter fragments of the tat protein containing the tat basic region (residues 49-57 having the sequence RKKRRQRRR. Barsoum et al. noted that moderately long polyarginine polymers (MW 5000-15000 daltons) failed to enable transport of .beta.-galactosidase across cell membranes (e.g., Barsoum on page 3), contrary to the suggestion of Ryser et al. (supra).
Other studies have shown that a 16 amino acid peptide-cholesterol conjugate derived from the Antennapedia homeodomain is rapidly internalized by cultured neurons (Brugidou et al., 1995). However, slightly shorter versions of this peptide (15 residues) are not effectively taken up by cells (Derossi et al., 1994).
The present invention is based in part on the applicants' discovery that conjugation of certain polymers composed of contiguous, highly basic subunits, particularly subunits containing guanidyl or amidinyl moieties, to small molecules or macromolecules is effective to significantly enhance transport of the attached molecule across biological membranes. Moreover, transport occurs at a rate significantly greater than the transport rate provided by a basic HIV tat peptide consisting of residues 49-57.